Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: A computational and experimental study Adel Hamza b , Yu Lan Piao c , Mi-Sun Kim a , Cheol Hee Choi c , Chang-Guo Zhan b , Hoon Cho a, ⁎ a Department of Polymer Science & Engineering, Chosun University, Gwangju 501-759, South Korea b Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA c Research Centre for Resistant Cells, Chosun University Medical School, Gwangju 501-759, South Korea abstract article info Article history: Received 7 June 2011 Received in revised form 10 August 2011 Accepted 30 August 2011 Available online 14 September 2011 Keywords: Alginate lyase AlyVI Homology modeling Docking Site-directed mutagenesis MD simulations The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure–affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k cat /K m ) of the double mutant L224V/D226G increased by two-fold com- pared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI struc- tures with improved catalytic activity. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Alginate is a copolymer of α-L-guluronate (G) and its C5 epimer β-D-mannuronate (M), arranged as homopolymeric G blocks, M blocks, alternating GM or random heteropolymeric G/M stretches [1]. It is a gelling polysaccharide common in the cell walls and intracel- lular material of brown seaweeds. It is also produced by two families of heterotrophic bacteria, Pseudomonadaceae and Azotobacteriaceae, often under strict regulatory control. Unlike the algae, these bacteria often produce polysaccharides substituted with O-acetyl groups on the 2 or 3 positions of D-mannuronate [2]. Their gelling ability, stabi- lizing properties, and high viscosity give alginates extracted from sea- weed wide use as stabilizers, viscosifiers, and gelling agents in foods and beverages, paper and printing materials, biomaterials, and phar- maceutical products [3]. Alginate hydrolysates have been shown to exhibit many important bioactivities, such as promoting the growth of Bifidobacteria spp., accelerating plant root growth, stimulating human keratinocytes, repressing the growth of HeLa cells, improving the function of β-lactogobulin, and enhancing the phagocytic activity of macrophages [3–5]. Alginates can be degraded by a group of en- zymes that catalyze β-elimination of the 4-O-linked glycosidic bond, forming unsaturated uronic acid-containing oligosaccharides. Therefore, alginate lyases (EC 4.2.2.3) are required in generating oligomeric algi- nates for research, analyzing alginate fine structure, and protoplasting red and brown algae [3,6] (Table 1). Alginate lyases have been used in the production of algal protoplasts and the study of alginate's fine structure with the aim of producing defined products for specific applications. Their cloning and sequencing have facilitated structure–function analyses of alginate lyases. Such analyses can aid the use of these enzymes in constructing important alginate polysaccharides. Alginate lyases have also been studied for the treatment of alginate polysaccharide buildup in the lungs of cystic fibrosis (CF) patients [7]. Alginate appears to be important in stabilizing biofilms formed by Pseudomonas aeruginosa and some other Pseudo- monas, with most Pseudomonas strains producing large amounts of extra- cellular alginates [8]. The high molecular mass and negative charge of bacterial alginate make it highly hydrated and viscous. Alginates com- prise the majority of the extracellular polymeric substance (exopoly- saccharide; EPS) of mucoid P. aeruginosa and have been reported responsible for the mechanical stability of biofilms and preventing antibiotic uptake [7,9]. Consequently, they are major pathogenic factors in CF patients [10]. Alginate lyase can remove EPS from the surface of mucoid Pseudomonas[11] and reduce the attachment of mucoid strains of P. aeruginosa[12]. For example, premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. This bacte- rium is ubiquitous and exhibits innate resistance to a wide range of antimicrobial agents, making infections both common and difficult to Biochimica et Biophysica Acta 1814 (2011) 1739–1747 ⁎ Corresponding author. Tel.: + 82 62 230 7635; fax: + 82 62 232 2474. E-mail address: hcho@chosun.ac.kr (H. Cho). 1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2011.08.018 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap